SignaIDT Biotechnologies (SZ), Inc. Dr. Lin Jingzhong published an article on China In Vitro Diagnostic Network (CAIVD), discussing the sensitivity of real-time fluorescent RT-PCR detection of new coronavirus from the perspective of primer probe design. The following is the full text.

After the outbreak of the novel coronavirus, early diagnosis has become the key to controlling the epidemic, and fluorescent PCR technology has become the first method of primary screening. At present, the detection rate of nucleic acid is only 30-40%, which makes the sensitivity of the detection kit attract much attention. In this issue, Dr. Lin Jingzhong's analysis of the sensitivity of the kit from the perspective of primer and probe design is recommended for readers.

 

I. Introduction

After the outbreak of the novel coronavirus, early diagnosis has become the key to controlling the epidemic, and fluorescent PCR technology has become the first method of primary screening. So far, several companies have obtained clinical registration approval for novel coronavirus fluorescent PCR detection reagents, and nearly a hundred companies have developed similar products. Recently, there have been many voices reflecting that the existing novel coronavirus fluorescent PCR detection kits have low sensitivity. The current detection rate is only 30-40% , and there have been many missed detections. The industry has conducted extensive discussions on this, and a consensus is that there are many reasons for the low detection rate and high false negative rate, including the sensitivity of the kit, the quality of the raw material of the kit, the efficiency of nucleic acid extraction, the quality of equipment, and operational errors. etc., but there is no systematic analysis of the reasons for the low sensitivity of the kit. The author believes that the design of primers and probes is the key to determining the sensitivity of the reagents. The problems in the design are inherent defects, which are difficult to be significantly improved by optimizing the components of the kit and adjusting the experimental conditions.

The sensitivity of early diagnosis is related to the success or failure of the prevention and control of the new coronavirus epidemic. At the same time, a large number of companies will apply for the registration approval of the product in the future, and finally push it to the testing market, which will have a profound impact on people's health. After the outbreak, the World Health Organization ( WHO ) published the sequences of novel coronavirus fluorescent PCR primers and probes designed by multiple countries. The author has been engaged in the research and development of molecular detection reagents, especially fluorescent PCR technology. After the outbreak of the novel coronavirus, we also carried out the research and development of related products, and we found that some of the primer probe designs from different countries issued by the WHO have obvious defects. This article takes the N gene primer probe designed by the US CDC as an example, discusses the basic principles of Taqman fluorescent PCR primer and probe design, and puts forward some suggestions for the reference of colleagues, avoiding detours and wasting resources, which can be regarded as doing our best to fight the epidemic. a force.

Any technology has advantages and disadvantages. From different perspectives, with different emphases, or with different parameters, algorithms or software, the conclusions may also be different. After all, theoretical analysis is not the same as the results of clinical sample testing. The sensitivity of the product has to go through clinical trials in the end, and meet the registration inspection requirements as the standard. The opinions in the article are inevitably biased, and criticisms and corrections are welcome.

 

2. The basic principle of Taqman probe fluorescent PCR

 

Real-time fluorescent PCR is a technique for real-time monitoring of PCR amplification of specific nucleic acid target sequences . Taqman probe method is the most widely used technique in real-time fluorescent PCR . Its basic principle is to add a pair of specific primers and a fluorescently labeled Taqman probe to the reaction. The 5' end of the probe is labeled with a reporter fluorescent group, and the 3' end is labeled with a quencher fluorescent group. When the probe is complete, due to the effect of fluorescence resonance energy transfer ( FRET ), the fluorescence of the reporter group is absorbed by the quencher group and emits a fluorescence that is different from the wavelength collected by the instrument, and the amplification signal cannot be detected, so the probe must rely on Taq The 5'-3' exo-cutting activity of the enzyme cuts the probe that has been combined with the template to form a double-strand after the primer is bound to the template with the process of double-strand synthesis, so that the reporter fluorescent group is separated from the quencher group . Shielded to emit the fluorescent signal that the instrument should detect. Commonly used reporter groups include HEX , FAM , ROX , JOE , VIC, etc., and quenching groups include TAMRA , BHQ , etc.

 

 

Three, Taqman probe method fluorescent PCR primer probe design

 

 Some of the most basic principles can refer to PrimerExpress 3.0 guideline (Table 1 ) of American ABI (Thermo Fisher) .

 

Table 1: Taqman fluorescent PCR probe and primer design guidelines (PrimerExpress 3.0, ABI)

TaqMan Probe (probe)	Primer _
The closer the distance between Probe and Primer , the better, the PCR product size is 50-150 bp
G/C%  is 30-80%
Avoid repeated sequences, especially avoid more than 4 consecutive G
Tm值: 68-70℃ (Quantification assay) 65-67℃ (Allelic Discrimination assay)	Tm value : 58-60 ℃
Probe  length : 13-25 bases (TaqMan MGB probe) 13-30 bases (TaqMan probe)	Primer  length : 20 bases (Optimal)
Avoid a sequence of 6 consecutive A 's	No more than 2 C+G in the first 5 sequences at the 3' end
The first column at the 5' end cannot be G ( if FAM-dye is selected , the second sequence at the 5' end cannot be G either)
Select the strand with more C than G as the probe
Avoid 3 or more Gs in the first 4 sequences at the 3 ' end (GGG-MGB-3'or GGAG-MGB-3')
Avoid the middle region of the probe containing 2 or more CC di-nucleotides

According to our experience, the most important parameters include position of primer probe, Tm value, hairpin structure, dimer. The N gene primer probe (Table 2 ) of the US CDC published by WHO will be discussed in detail below .

 

Table 2: Primer probe sequences designed by US CDC 

 

 

01 Position of primer probe

In the design principles of primer probes, the most important thing is to avoid false negatives (missed detections) while ensuring specificity (avoiding false positives), so it is necessary to select conservative (that is, avoid mutations as much as possible) within the group (sequences to be detected). or using degenerate sequences), between groups (control sequences) specific regions. In addition, in order to obtain the best amplification effect, the basic requirements in Table 1 should also be met as far as possible.

Using the N gene sequence of SARS and bat CoV as a control, the N gene sequence of the new coronavirus was sorted. It can be seen from Figures 1 , 2 , and 3 that in the design of the US CDC , except that the second set of upstream primers is not specific, The positions of the rest of the probes and primers are selected from the specific regions between the groups that are conserved within the group. The second set of probes and downstream primers are in specific regions, so non-specific amplification will not occur.

Figure 1: Location of the first set of primers and probes for 2019-nCoV from the US CDC

Figure 2: US CDC 2019-nCoV second set of primer probe positions

 

Figure 3: Location of the third set of primers and probes for 2019-nCoV from the US CDC

 

The third set of designed probe N3P is not specific to 2019-nCoV (can bind to bat CoV at the same time ) , and the second position at the 5' end is set to Y , that is, T/C degeneracy, which just cannot distinguish between new coronavirus and bat coronavirus and SARS virus. However, the upstream and downstream primers are specific to 2019-nCoV and may not produce non-specific (false positive) amplification.

02 Tm value

Taqman probe fluorescent PCR usually adopts a two-step method, that is, denaturation at about 94°C , annealing and extension at 60°C . Therefore, usually the Tm value of the primer is designed at 60±2°C , and the probe is at 70±2°C .

 

Figure 4: Tm values ​​of the first US design

 

Figure 5: The second set of Tm values ​​for USCDC N genes

 

Figure 6a: The third set of Tm values ​​of the USCDC N gene, the second sequence at the 5' end of the probe is C

 

Figure 6b: The third set of Tm values ​​of the USCDC N gene, the second sequence at the 5' end of the probe is T

 

It can be seen from Figures 4, 5, and 6 that the Tm values ​​in these three designs basically meet the requirements in Table 1. The second sequence at the 5' end of the probe N3P in the third set is designed as Y (T/C degeneracy), when it is C here (Figure 6a), the Tm value of the probe N3P is slightly higher, but it should not be through optimization become the main problem.

03 Hairpin structure of primers and probes

Stable secondary structures (dimers or hairpins) are important factors that reduce the utilization efficiency of primers and probes, especially the hairpins of probes, which can easily lead to a decrease in probe utilization, resulting in low fluorescence signals and affecting detection sensitivity. Generally, the dG of the hairpin should be controlled ≥ -1.0 (Note: dG is a parameter used to measure the energy required for cleavage of dimers or hairpins. The more stable the secondary structure, the more energy required for cleavage, and the smaller the dG) .

Using DNA Star software to analyze the hairpin structure of the three sets of primer probes designed by the US CDC, the results (Figure 7) found that the first set of downstream primer N1R has a stable hairpin structure (dG=-2.3kc/m ), the third set of probe N3P has a slightly stable hairpin structure (dG=-1.3kc/m), and the rest of the primers and probes have no stable hairpin.

 

Figure 7: Hairpin structures of USCDC primers and probes

 

04 dimer

During the design, the dimer (self dimer or paired dimer) is usually controlled at dG>-5.0kc/m, preferably dG>-3.6 kc/m. It can be seen from the self-dimers of the three sets of primer probes shown in Figure 8 that the second set of probes has a very stable self-dimer (dG=-13.1kc/m), and the upstream primer N2F of the second set has A stable self-dimer (dG=-9.3kc/m), the third set of downstream primers has a relatively stable self-dimer (dG=-7.0kc/m).

Figure 9 shows that the US CDC 's second set of primer probes has relatively stable paired dimers ( dG=-9.0kc/m ). Figure 10 shows that the third set has two relatively stable paired dimers ( dG=-10.1kc/m ).

Figure 8: Self-dimerization of US CDC gene primers and probes

 

Figure 9: Paired dimers of the US CDC’s second set of primer probes

 

Figure 10: USCDC third set of primer-probe paired dimers

 

In addition, when using one tube for multiple detection (multiple PCR ), that is, when one tube detects multiple sites or multiple pathogens at the same time, the paired dimers of primers and probes between different sites should also be considered. For example, when the first set and the second set are combined (Figure 11 ), the forward primers of the first set and the probes of the second set have a relatively stable paired dimer ( dG=-8.3kc/m ). When the first set and the third set are combined (Figure 12 ), the probes of the first set and the forward primers of the third set have a stable dimer ( dG=-12.2kc/m ). When the second set and the third set are combined, the upstream primers of the second set and the upstream and downstream primers of the third set each have a relatively stable dimer ( dG=-7.0kc/m Figure 13 ).

 

Figure 11: The first and second sets of paired dimers of the US CDC N gene

  

Figure 12: The first and third sets of paired dimers of the US CDC N gene

 

Figure 13: The second and third sets of paired dimers of the US CDC N gene

 

To sum up, the three sets of primers and probes designed by CDC in the United States, the selection of position and the design of Tm value are relatively reasonable, but the secondary structure is relatively stable. The reverse primer N1R of the first set has a very stable hairpin ( dG=-2.3kc/m , Figure 7 ). The self-dimer of the second set of probes is very stable ( dG=-13.1kc/m , Figure 8 ), and the self-dimer of the upstream primer is also relatively stable ( dG=-9.3kc/m , Figure 8 ), relatively stable There are more paired dimers ( dG=-6.8 ～9.0kc/m, Figure 9 ), and the utilization efficiency of primers and probes will be relatively low. The third set of probes, N3P, has a relatively stable hairpin ( dG=-1.3kc/m , Figure 7 ), and both probes and primers have relatively stable paired dimers (Figure 10 ).

 

4. Judgment of results

The CDC of the United States puts forward its judgment on the results, and believes that only when the simultaneous amplification curves of the three sites exceed the threshold value can it be judged as positive (Figure 14 ).

The author believes that this judgment has no theoretical basis. In addition to a pair of specific primers, Taqman probe fluorescent PCR also has a specific probe, and the theoretical probability of non-specific amplification based on the design is very small. In the detection of new coronaviruses, take the first set of primer probes from the CDC in the United States as an example. It can be seen from Figure 1 that there is a 3-bp insertion in the upstream primer for the SARS virus, which is impossible to amplify. There are 4 mutations, 1 mutation in the probe, and 5 mutations in the downstream primer . In theory, the probability of detecting the closest bat sequence (false positive) due to mismatch is (1/4) ¹⁰ =9.5367x10 ^-7 . If a false positive occurs, the most likely cause is cross-contamination or aerosol contamination. The correct judgment should be that the amplification curve of at least one of the above three sites exceeds the threshold value, and the case can be judged as positive. Most of the fluorescent PCR detection reagents currently on the market only use one site.

 

Figure 14: US CDC guidelines for judging real-time fluorescent RT-PCR results

V. Conclusions and Recommendations

In the design principles of primer probes, the most important thing is to avoid false negatives (missed detections) while ensuring specificity (avoiding false positives), so it is necessary to select conservative (that is, avoid mutations as much as possible) within the group (sequences to be detected). or use degenerate sequences), between groups (control sequences) specific regions, while maximizing the utilization efficiency of primers and probes in the reaction. The author believes that the importance of the parameters is the position of the primer probe, the hairpin structure of the probe, the relative position of the probe to the upstream primer, the Tm value, the primer hairpin, and the dimer.

The author made a detailed analysis of the primers and probes designed by seven countries published by WHO, and found that there are certain defects, some of which are more obvious, mainly reflected in: 1) non
- specific ( E gene in Germany, Orf1b gene in Hong Kong University) ;
2 ) The hairpin structure of the probe is stable (the probe of the Chinese CDC Orf1ab gene, the German SARS RdRP gene probe P1 , the probe of the N
gene of the University of Hong Kong); 3 ) The relative position of the primer and the probe is unreasonable (China CDC 's N gene, Germany's E gene, Hong Kong University's Orf1b gene and E gene (designed on the reverse chain), Japan's N gene);
4) Tm value is unreasonable ( N gene and Orf1ab gene of CDC in China, SARS RdRP gene and E gene in Germany , Orf1b gene and N gene in Hong Kong University , N gene in Japan , N gene in Thailand );
5 ) Hairpin of primer Structural stability ( the first set of reverse primers for the CDC N gene in the United States, the forward and reverse primers for the Orf1b gene of the University of Hong Kong, and the forward primer for the N 
gene of the University of Hong Kong); 6 ) The self-dimer stability of the primer probe (China CDC Orf1ab gene reverse primer, American CDC N gene second set of probe N2P , German E gene probe P1 , Hong Kong University N gene forward primer and probe); 7
) paired dimerization of primers and probes body stability (ChinaN gene of CDC , second and third sets of N gene of the United States, E gene of Germany ).

The author believes that probes with stable hairpin structures are basically unusable. The amplification efficiency of the probe that is too far away from the upstream primer is low, it is difficult to improve the efficiency through optimization, and should be avoided as much as possible. Other secondary structures may be improved by adding PCR accelerators or adding excess components (primers, probes, Taq enzymes, etc.), and the unsatisfactory Tm value can be adjusted by adjusting the temperature of annealing and extension, but like some of the above designed primers and probes Needle Tm values ​​that are almost the same are unreasonable and should be avoided.

In fact, the genome of the new coronavirus differs from the closest bat coronavirus by 4% , and from the SARS virus by 20% , and the current data shows that the differences within the Wuhan new coronavirus are very small, about 100,000 points The third ( 3.3x10 ^-5 ), there are many regions in the genome or in the N gene and Orf1ab gene can design specific, high-efficiency primer probes for the development and application of fluorescent PCR detection reagents.

There is no real shortcut in the face of science and technology . Scientific design of primers and probes isthe basis for the development of fluorescent PCR detection reagents and the most basic element to ensure the sensitivity and specificity of the kit. In order to avoid detours and waste of resources, it is recommended that manufacturers in the industry develop new coronavirus fluorescent PCR detection reagents in strict accordance with the principles of fluorescent PCR technology primer probe design.

About the author: Lin Jingzhong, Ph.D., University of Toronto, Canada, postdoctoral fellow, University of California, USA. Shenzhen overseas high-levelB-class talents. He is currently the chief scientist/general manager of Shenzhen Segno Biotechnology Co., Ltd. He used to be the founder/general manager of Shenzhen Taitai Gene Engineering Co., Ltd., and a senior researcher of Lynx Pharmaceutical Company in the United States.

Contributed and edited: Yi Shui | Proofreading: Bonnie  | Editor in charge: Sun Xuguang